Understanding the interactions between macromolecules is a central theme of biology, with complementarity in the surface character and shape of these molecules usually defining both the specificity and strength of their interactions. Traditionally, the best method of precisely defining these contact surfaces is to determine the tertiary structure of an interacting complex by X-ray diffraction or by multi-dimensional NMR techniques. However, these approaches are not always technically feasible, are very costly, and can be time consuming. Easier methods for helping to define interacting surfaces at the molecular level could prove extremely useful, for instance in the exploration of protein-protein contacts involved in receptor/ligand interactions, in understanding the basis of enzyme/substrate specificities, and in the mapping of antibody epitopes, to name just a few examples. However, a formidable obstacle to be overcome in the development of such new techniques is the tremendous structural diversity of biological macromolecules.
Recently a method has been successfully pioneered by Smith and others (Smith, Science 228:1315-1317 (1985); Scott et al., Science 249:386-390 (1990); and Parmley et al., Gene 73:305-318 (1988)) that enables screening of huge populations of diverse macromolecules, and selecting specific members of these populations on the basis of their binding affinity to an immobilized protein target molecule. In this technique, termed the phage-display method, DNA sequences encoding highly diverse libraries of short peptides are fused to the 5'-ends of bacteriophage coat protein genes. Following expression, these fusions are correctly folded and assembled, exposing the random peptides on the bacteriophage surface. The phage/peptide libraries are then given the opportunity to bind to a target immobilized protein, typically a monoclonal antibody, and phage displaying peptides that interact specifically with the target are selectively retained through a washing procedure. The retained phage particles are then eluted for additional rounds of selections or for analyses.
Since its introduction, the phage-display technique and its variations have been applied to map a wide range of protein-protein or protein-ligand interactions (Djojonegoro et al., BioTechnology 12:169-172 (1994); Oldenberg et al., Proc. Nat'l. Acad. Sci. U.S.A. 89:5393-5397 (1992); Scott et al., Proc. Nat'l Acad. Sci U.S.A. 89:5398-5402 (1992); Blond-Elguindi et al., Cell 75:717-728 (1993); and Hammer et al., J. Exp. Med. 176:1007-1013 (1992)). The peptide sequence information derived from these studies is useful, but the ability to perform structural studies on the peptides obtained is limited both by the low expression levels of phage coat protein genes and by the character of the peptides selected by these systems, which are usually unconstrained molecules possessing many degrees of conformational freedom. This structural flexibility renders difficult any subsequent structural studies on these molecules.
Also of background interest to the present invention is Stahl et at., U.S. Pat. No. 4,801,536; issued Jan. 31, 1989; incorporated by reference, which disclose that C-terminal fusions of peptides and proteins to flagellin can be made and exported from the cell. Unfortunately, such fusions do not assemble into functional or even partially functional flagella. McCoy et al., U.S. Pat. No. 5,292,646; issued Mar. 8, 1994; incorporated by reference, discloses that both N- and C-terminal fusions of peptides and proteins to thioredoxin can be made. However, these fusion proteins reside in the bacterial cytoplasm, i.e., on the interior of the cell. Furthermore, while fusions of a wide variety of peptide sequences were shown by McCoy et at., supra, to be permissible into the active-site loop of thioredoxin and without deleterious effects on thioredoxin protein folding; nevertheless, these active-site loop fusions also reside in the bacterial cytoplasm.
Accordingly, there continues to be a need in the art for alternative methods and reagents which address these problems.